EP1603678B1 - Methods and devices for separating particles in a liquid flow - Google Patents

Abstract

Methods and devices for the separation of particles (20, 21, 22) in a compartment (30) of a fluidic microsystem (100) are described, in which the movement of a liquid (10) in which particles (20, 21, 22) are suspended with a predetermined direction of flow through the compartment (30), and the generation of a deflecting potential in which at least a part of the particles (20, 21, 22) is moved relative to the liquid in a direction of deflection are envisaged, whereby further at least one focusing potential is generated, so that at least a part of the particles is moved opposite to the direction of deflection relative to the liquid by dielectrophoresis under the effect of high-frequency electrical fields, and guiding of particles with different electrical, magnetic or geometric properties into different flow areas (11, 12) in the liquid takes place.

Description

The present invention relates to methods for separating particles in a fluidic microsystem, in particular under the action of electrophoresis, and fluidic microsystems adapted to carry out such methods.

In the biomedical and chemical-analytical technique, the separation of micro-objects such. As particles of natural or synthetic origin or molecules in fluidic microsystems under the action of electrically or magnetically induced forces increasingly important. Two conventional separation principles, which basically differ according to the type of electrical separation forces, are illustrated schematically in FIGS. 10A, B.

Figure 10A shows schematically the separation by means of negative dielectrophoresis (see, for example, DE 198 59 459). In a fluidic microsystem 100 ', particles having different dielectric properties flow through a first channel 30'. By means of an electrode arrangement 40 ', a field barrier extending transversely across the channel 30' is generated by application of high-frequency electric fields which, depending on the dielectric properties of the particles, has a permeable or laterally deflecting effect in cooperation with the flow forces. Particles 22 'having a low dielectric constant (or conductivity) compared to the medium are deflected into an adjacent channel 30A', while particles 21 'having a higher dielectric constant (or conductivity) continue in channel 30'. Since the dielectrophoresis depends on the particle size (see T. Schnelle et al., In "Naturwissenschaften" Bd., 83, 1996, pp. 172-176), even with the same dielectric Characteristics of a separation of the particles made according to the size. Conventional dielectrophoretic particle separation may have disadvantages in terms of separation reliability, especially for particles with similar dielectric constants, and the complexity of the channel design. The reliability of the separation may be limited especially in the separation of biological cells of the same type into different subtypes (eg macrophages, T lymphocytes, B lymphocytes).

Another problem that has only been solved to a limited extent in conventional dielectrophoretic particle separation can be the occurrence of undesired cell constituents in biological suspension samples. Cell constituents often can not be distinguished from complete cells solely by their dielectrophoretic properties. Furthermore, in microsystems they can lead to unwanted accumulations of channel constrictions and blockages to system failure. Finally, unwanted cell constituents can also interfere with measurements on cells, such as a patchclamp measurement. There is therefore an interest in an improved method for purifying suspension samples, which has a higher reliability than the dielectrophoretic particle separation.

Figure 10B illustrates an electrophoretic separation of particles, e.g. Molecules in a microstructured channel (see T. Pfohl et al., In "Physik Journal", Vol. 2, 2003, pages 35-40). At the ends of the channel 30 'formed alternately with wide and narrow sections, electrodes 41', 42 'are arranged, which form an electrophoresis field when subjected to a DC voltage in the channel 30'. The drift velocity of the molecules in the electrophoresis field depends on their molecular weight and charge. In the wider sections of the channel 30 ', the drift velocity of the larger molecules is lower, so that in the course of the separation first the small molecules and later the large molecules arrive at the end of the separation distance. Although the electrophoretic separation in fluidic microsystems has the advantage that it can be dispensed with the use of a separation gel as in macroscopic electrophoresis. However, the principle shown in FIG. 10B has the disadvantage that a separate microsystem with adapted geometrical parameters must be provided for each separation task and in particular each particle type. Another disadvantage is that the separation takes place in the dormant liquid, because this is associated with a high expenditure of time and additional measures for adaptation to flow systems.

The separation principles mentioned above are also mentioned in WO 98/10267. In the channel of a fluidic microsystem charged particles z. B. electrophoretically pulled from a sample in a parallel flowing buffer solution. This technique is limited to samples with certain characteristics of the sample components. It is also disadvantageous because the particles can be electrophoretically drawn to the channel walls, which in particular in biological materials, eg. B. cells is undesirable.

The electrophoretic deflection of particles is also described in DE 41 27 405. Particles are moved in a quiescent fluid under the action of traveling electrical waves. If they pass by electrophoresis electrodes during the movement, a separation takes place according to the electrical properties of the particles. There are the same disadvantages as in the o. G. WO 98/10267.

It is also known to combine dielectrophoretic and electrophoretic field effects in the manipulation of particles in fluidic microsystems. According to DE 195 00 683, liquid-suspended particles are in an electrode arrangement held, which forms a closed field cage (potential well) when exposed to high-frequency AC voltages by negative dielectrophoresis. In order to correct positional variations due to thermal shocks in precision measurements, particles in the field cage are additionally electrophoretically displaced. The electrophoretic displacement takes place in the context of a control loop depending on the example, optically determined position variations of the particle. The technique described in DE 195 00 683 is not suitable for particle separation because it represents a closed, stationary measuring system. Furthermore, the combination of dielectrophoresis and electrophoresis on the closed field cage is limited to relatively large, single particles. Disadvantages may arise in the measurement of, for example, macromolecules, since in these the effect of the negative dielectrophoresis is significantly lower than that of the electrophoresis, so that it can lead to an undesired attachment of the macromolecules to the electrodes. Particle groups can not be measured with this technique, since all particles require their own correction movement. Separation of particles would also be hampered by a dipole-dipole effect (see T. Schnelle et al., "Naturwissenschaften", Vol. 83, 1996, pp. 172-176), which promotes particle aggregation.

DE 198 59 459 also discloses the combination of alternating and direct voltages in fluidic microsystems for targeted cell fusion or fusion. In this technique, the effect of the DC voltage on the fusion or poration is limited, a particle separation is not provided.

From the publication by S. Fiedler et al. in "Anal Chem." Vol. 67, 1995, p. 820-828, it is known to generate by a possibly pulsed DC voltage control of microelectrodes in aqueous electrolyte solutions temporal or local, detectable with fluorescent dyes pH gradients.

For pharmacological, analytical and biotechnological research is not only an interest in a separation of particle mixtures according to geometric (size, shape) or electrical properties (dielectric constant, conductivity), but also according to other parameters, such. As surface charges or charge-volume ratios. The occurrence of surface charges is described, for example, by N. Arnold et al. in "J. Phys. Chem." Vol. 91, 1987, pp. 5093-5098, L. Gorre-Talini et al. in "Phys. Rev. E" Vol. 56, 1997, pp. 2025-2034 and Maier et al. in "Biophysical J." Vol. 73, 1997, pp. 1617-1626.
Document WO 00/00292 discloses the method of separation according to the preamble of claim 1 and a fluidic microsystem according to the preamble of claim 21.

The object of the invention is to provide improved methods for the separation of particles in liquid flows in fluidic microsystems, with which the disadvantages of conventional techniques are avoided. Processes according to the invention are to be distinguished, in particular, by a broader field of application with a multiplicity of different particles and increased reliability in particle separation. The object of the invention is also to provide improved microsystems for implementing such methods, in particular improved microfluidic separation devices, which are characterized by a simplified construction, a high reliability, a simplified control and a wide range of applications for various particles.

These objects are achieved by methods and devices having the features of claims 1 and 21. Advantageous embodiments and applications of the invention will become apparent from the dependent claims.

The present invention is based on the general technical teaching of the method and apparatus, at least one particle suspended in a liquid by means of a combined exercise of separation forces, on the one hand focusing dielectrophoretic separation forces and on the other hand distracting separation forces, such as electrophoretic separation forces to move in a state of continuous flow within the liquid, so relative to the flowing liquid. The at least one particle can be directed into at least one separating device in the fluidic microsystem, depending on its geometric, electrical, magnetic or derived properties in a certain flow range during the pre-accession. Depending on the orientation of the deflecting separation forces (deflection direction) relative to the direction of movement of the liquid (flow direction), the flow region may comprise a specific flow path within the flow cross-section of the liquid or a downstream or downstream portion of the flow.

The movement of the particle into a particular flow area allows separation of particle mixtures during the continuous flow of the particle suspension, for example, through a group of multiple electrodes. The release effect is based on the specific reaction of different particles to the different deflecting and focusing field effects. In contrast to the separation at field barriers, a separation distance can be traversed, whereby the reliability of the targeted movement of individual particles can be increased, for example, to specific, preferably two, flow paths. The effect of the electric fields can be tuned by adjusting the field characteristics (in particular frequency, voltage amplitudes, clock, etc.) to the parameters of the particles to be separated. The invention enables a simplified construction of the electrophoretic separator, since no gels for embedding electrophoresis electrodes or special channel shapes are needed. Furthermore, a gas formation by suitable control of the electrodes in combination with the permanent Flow can be avoided. The invention also has advantages particularly in terms of reliability and selectivity in particle separation into different flow paths, and high efficiency and high throughput of separation.

According to the invention, a separation of particles in a compartment, in particular a channel of a fluidic microsystem, through which particles flow in the suspended state, wherein at least a portion of the particles or particles of at least one type under the action of a deflecting potential from the sample to be separated in a predetermined deflecting direction (first reference direction, for example to the edge of the compartment) are moved to the effect that simultaneously or temporally and / or spatially alternating under the action of an opposite potential by dielectrophoresis, in particular negative or positive dielectrophoresis an opposite movement of the particles (second reference direction , for example, away from the walls or as a rally in the canal center). Advantageously, particles with different electrical, magnetic or geometric properties experience the potential effects as separation forces in various ways, so that different effective forces (potential minima) form due to the combined application of the potentials to which the particles migrate. The potential minima are z. B. in the flow cross-section of the liquid, so that a separation in the flow to different flow paths is possible. The focusing, dielectrophoretic potential is preferably formed towards the center of the channel acting. If, in the channel cross-section, the electrodes are arranged essentially on a circular line, the focusing potential with respect to the flow direction in the channel can advantageously be formed radially symmetrically.

The particles which are preferably separated or separated from one another by the technique according to the invention generally comprise colloidal or individual particles with a diameter of z. B. 1 nm to 100 microns. There may be synthetic particles (eg, latex beads, superparamagnetic particles, vesicles), biological particles (eg, cell groups, cell constituents, cell debris, organelles, viruses) and / or hybrid particles made of synthetic and biological, different synthetic or different are constructed biological particles, subjected to the separation process of the invention.

Advantageously, the electrophoretic mobility μ ( v = μ · E ) for cells depends not only on the composition of the outer medium, ie the suspension liquid (in particular conductivity, ion composition, eg Ca ²⁺ content and pH), but also cell type so that different cell types or within a cell group of the same cell types different subtypes (eg., Macrophages, T lymphocytes, B lymphocytes) can be differentiated with the inventive technique within a cell group. The distinction of the subtypes represents a particular advantage of the invention, since these are poorly distinguishable with conventional dielectrophoretic separation methods. By combining a dielectrophoretic focusing according to the invention, the selectivity is increased especially for cells of the same type.

If the particles to be separated a mixture of biological cells and cell components, such as. As cell debris, the separation method can be advantageously used for cleaning a suspension sample with suspended biological material. The material which, for example, is inhomogeneously composed after cultivation and, for example, complete cells, dead cells, living cells or fragments of cells, such as, for example, cells. As organelles, cell residues or protein clumps can be purified by the method according to the invention. The Unwanted fragments of cells can be removed from the microsystem via certain flow paths. An adverse effect on the following structural elements in the microsystem, such. As a clogging of channels by cell components can be avoided.

Advantageously, the deflecting potential can be generated by electrical, magnetic, optical, thermal and / or mechanical forces and thus adapted to a wide variety of applications and particle types. Mechanical forces include, for example, forces transmitted by sound, additional currents or inertia. The deflecting potential can in particular be given by a gravitational field, wherein according to the invention the movement of the particles in the focusing potential (by high-frequency electric fields) is superimposed with a sedimentation movement of the particles.

If, according to a preferred embodiment of the invention, the deflecting separation forces comprise electrical forces under the effect of which the particles are drawn by electrophoresis from the liquid towards the edge thereof, there may be advantages in terms of the separation result. The combination of electrophoresis and dielectrophoresis for particle separation can in particular have advantages in the separation of biological materials which, for example, react very differently to electrophoresis and dielectrophoresis depending on the material or particle size and can therefore be separated with high selectivity.

Advantageously, the DC fields for the electrophoretic particle movement according to a further embodiment of the invention can additionally be used for an electrical treatment of the particles. It is known that biological cells can be lysed in static electric fields. Lysis involves an electrically induced change, for example destruction of the cells. The lysis serves For example, the preparation of cell material for PCR procedures. Since the effect of the lysis is field-strength-dependent, it is provided according to a particularly preferred embodiment of the invention that certain cells are deflected from a cell mixture by electrophoresis in a flow region near the electrodes, where due to the smaller distance from the electrodes, the field strength is higher and thus the lysis is carried out simultaneously to the process of particle separation.

The selectivity can also be flexibly adjusted by a suitable AC voltage control. By changing the phase position of fields, the dielectric potential can be shaped differently in the case of negative dielectrophoresis. In addition, can be impressed by the DC voltage control pH profiles that affect the electrophoretically or dielectrically effective potential.

In the combination of electrophoresis and dielectrophoresis according to the invention, the separating devices for generating the opposing potentials can advantageously be formed by a common unit. The separator comprises electrodes which are disposed on walls of the channel and which are supplied with electric fields for the production of dielectrophoresis and electrophoresis. Advantages for the control of the separation may in particular arise if the electric fields comprise high-frequency AC components and DC components which are generated simultaneously or alternately.

According to a modified variant of the invention, the deflecting separation forces may comprise electrical forces which, like the focusing potential, are generated by high-frequency electric fields. The deflection can thus also be generated by suitable dielectrophoretic forces formed by high-frequency electrical signals, z. B. sine or square wave signals are superimposed with suitable frequency components.

According to a preferred embodiment of the invention, the deflecting and focusing potentials may be formed alternately in time in at least a portion of the channel. On average, the particles effectively have a potential that corresponds to the superposition of both potentials. Advantageously, thus the control of the at least one separating device can be simplified.

According to a further preferred embodiment of the invention, the two potentials can be generated alternately in different, successive sections of the channel. Advantageously, thus the structure of the microsystem can be simplified.

It may be particularly advantageous for the maintenance of the separation result if the flow paths open into further, separate compartments of the microsystem. If the separated fractions have flowed into the adjoining compartments, a subsequent mixing is excluded. This separation of the fractions may be particularly effective if the compartments are separated by channel walls or electric field barriers.

According to a further embodiment of the invention it can be provided that in the compartments a further separation according to the principle of the invention, for example, a combined exercise of electrophoretic and dielectrophoretic field effect takes place. Thus, advantageously hierarchical separation principles can be realized with a separation in coarse and subsequently in fine fractions. However, the sequence of several separation processes in the manner of a cascade into different fractions is not necessarily to the provision bound to separate compartments. Rather, the realization of the separation cascade with flow paths in a common, sufficiently wide channel of the microsystem is possible.

According to a modification of the invention, the flow in the microsystem can be directed so that particles pass through a separation stage several times, so that advantageously the separation result can be improved even more.

Further advantages of the invention may result if, after the separation (deflection into different flow areas), a detection takes place in the flow areas for checking the separation result. The detection comprises, for example, a known optical measurement (fluorescence measurement or transmitted light measurement) or a known impedance measurement.

Advantageously, depending on the measurement result, for. B. depending on the quality of separation or occurring faulty separations, the control parameters of the deflecting and focusing potentials are adjusted so that improves the separation effect.

The effectiveness of the separation according to the invention can advantageously be increased if the particles first pass past a dielectrophoretic or hydrodynamic line-up element. At this, individual particles or a group of particles are strung on a particular flow path on which they pass at the separators, for example, the electrodes for performing dielectrophoresis and electrophoresis.

If, according to a further variant of the invention, a pH gradient is generated in the channel of the microsystem in which the particle separation takes place, advantages for the separation effect can result. Due to the pH gradient, the effect of the distracting Potentials such. B. the electrophoretic cell particle movement location-dependent. This allows a particle deflection into different flow paths as a function of the particle position along the flow direction through the channel. Advantageously, a particularly simple design of the microsystem results when the pH gradient is generated electrochemically using the electrodes, which are also used to form the DC field for electrophoresis.

Another advantage of the invention is that the particle separation can take place simultaneously in several spatial directions. According to the invention, a plurality of deflecting potentials with different effective directions can be generated with the focusing potential, which is then preferably acting towards the center of the channel, in order to simultaneously separate the particles to be separated with respect to two different features, such as e.g. B. to separate dielectric and magnetic properties.

Another object of the invention is a fluidic microsystem, which is adapted to implement the method according to the invention and in particular comprises at least one separating device for exercising focusing dielectrophoretic separation forces and deflecting separation forces. A fluidic microsystem with at least one compartment, for example a channel for receiving a flowing liquid with suspended particles and a first separator for generating a deflecting, the particles in the first reference direction, for example, from the center of the flow pulling potential is in particular with a second separator equipped for generating at least one focusing, opposite potential. Under the action of high-frequency electric fields, the particles with the second separator become by dielectrophoresis from the lateral Walls of the channel and / or disposed thereon electrodes or other parts of separating devices repelled.

According to a preferred embodiment of the invention, the first separating device is designed to generate electrical, magnetic, optical and / or mechanical forces. It comprises, for example, an electrode device with electrodes or electrode sections and in this case forms a common deflection unit with the second separation device. Alternatively, the first separator comprises a magnetic field device, a laser or an ultrasound source. These components are inventively combined for the first time for the separation of flowing particles with a dielectrophoretic manipulation.

If the separating devices form a common deflection unit, a simplified structure of the microsystem advantageously results. The deflection unit preferably comprises electrodes, which are constructed like micro-electrodes known per se in fluidic microsystems. The electrodes can be controlled alternately in time.

The electrodes for combined dielectrophoresis and electrophoresis are preferably arranged on insides of the walls of the compartment. In this design, there may be advantages in terms of the effectiveness of the field effect.

Since the separation devices can act alternately or temporally and / or spatially alternately, so that particles are directed to different flow paths depending on the effective time potentials, it is advantageously possible for the first and second separation devices to be separate in different, consecutive Sections of the compartment are arranged. The separation devices comprise, for example, electrode sections which can each be activated for dielectrophoresis or electrophoresis.

Figur 1:

eine schematische Draufsicht auf eine erste Ausführungsform eines erfindungsgemäßen Mikrosystems (Ausschnitt),

Figur 2:

eine Querschnittansicht des Mikrosystems gemäß Figur 1 entlang der Linie II-II,

Figur 3:

eine Querschnittansicht des Mikrosystems mit schematisch illustrierten Potentialverhältnissen,

Figuren 4

bis 7: schematische Draufsichten auf weitere Ausführungsformen erfindungsgemäßer Mikrosysteme (Ausschnitt), und

Figur 8:

eine schematische Querschnittsansicht einer Elektrodenanordnung zur Illustration einer Ausführungsform der Erfindung, bei der mehrere ablenkende Potentiale erzeugt werden,

Figur 9:

eine Kurvendarstellung zur Erklärung der Erzeugung eines ablenkenden Potentials durch die Überlagerung dielektrophoretischer Kräfte,

Figuren 10A, B:

schematische Illustrationen herkömmlicher Mikrosysteme mit einer dielektrophoretischen (A) und einer elektrophoretischen (B) Trennung.

Further details and advantages of the invention will be described below with reference to the accompanying drawings.
Show it:

FIG. 1:

a schematic plan view of a first embodiment of a microsystem according to the invention (detail),

FIG. 2:

FIG. 2 shows a cross-sectional view of the microsystem according to FIG. 1 along the line II-II, FIG.

FIG. 3:

a cross-sectional view of the microsystem with schematically illustrated potential ratios,

FIGS. 4

to 7: schematic plan views of further embodiments of inventive microsystems (detail), and

FIG. 8:

3 is a schematic cross-sectional view of an electrode arrangement illustrating an embodiment of the invention in which a plurality of deflecting potentials are generated;

FIG. 9:

a graph to explain the generation of a deflecting potential by the superposition of dielectrophoretic forces,

FIGS. 10A, B:

schematic illustrations of conventional microsystems with a dielectrophoretic (A) and an electrophoretic (B) separation.

The invention will be described below with reference to the separation of particles in the channel of a fluidic microsystem. Fluidic microsystems are known per se and are therefore not described in further detail. The implementation of the invention is not restricted to the illustrated channel structures, for example in chip structures or in hollow fibers, but can generally also be implemented in differently shaped compartments.

The combination according to the invention of focusing and deflecting forces whose superposition for the particles to be separated lead, depending on the particle properties, to different equilibrium positions (flow paths or sections) in the liquid flow, with two separation devices or a combined separating device, will be described with reference to the preferred embodiment Combination of dielectrophoresis and electrophoresis described. If the deflecting force has at least one vector component in a reference direction (deflection direction) perpendicular to the direction of fluid movement in the channel, the dielectrophoresis acts from the walls of the channel into the interior of the flow cross-section of the flowing fluid, while the electrophoresis reverses to the outer edge of the fluid Flow profile, in particular to electrodes on the walls towards directing effect. Analogous to the principles explained below, other deflecting forces can be used. On the other hand, when the deflecting force is parallel to the direction of fluid flow, the dielectrophoresis acts to focus along the fluid flow, modulating the dielectrophoretic effect to move the particles in the electrophoresis field at different rates.

Figures 1 and 2 show a detail of an inventive fluidic microsystem 100 in an enlarged schematic plan view and cross-sectional view. The microsystem 100 includes a channel 30 bounded by the lateral channel walls 31, 32, the channel bottom 33 (top view in FIG. 1) and the top surface 34. On the channel bottom 33 and the top surface 34 electrodes 40 are formed as a separator. Furthermore, funnel electrodes 51, 52 of a dielectric alignment element 50 are provided. The structure of the microsystem 100 and the formation of the electrodes and their electrical connection are known per se from microsystem technology. The channel has, for example, a width of approx. 400 μm and a height of approx. 40 microns (these ratios are not shown to scale in the figures). The lateral electrode spacing in the planes of the channel bottom 33 and the top surface 34 is, for example, 70 μm, while the vertical distance between the opposing electrodes corresponds to the channel height rd. 40 microns.

The electrodes 40 comprise straight electrode strips extending longitudinally of the channel 30, i. extend in the flow direction through the channel. The electrodes 40 are divided into individual electrode segments 41, 42,... In each case a group of electrode segments forms an electrode section, which can be controlled separately. Each segment has a width of about 50 microns and in the flow direction a length of z. B. 1000 microns. Each electrode section is connected to a controller 70 (shown here only for the electrodes 41, 42).

The control device 70 is designed to act on the electrodes 40 with voltages such that the passing particles in an electrode section (for example 45-48, see FIG. 2) are exposed to repulsion from the electrodes by means of negative dielectrophoresis and / or electrophoretic drift motion perpendicular to the flow direction become. The controller includes an AC generator 71 and / or a DC generator 72 connected to the electrodes. The AC generator 71 may be equipped with an actuator, with which the amplitudes of high-frequency AC voltages can be adjusted at the electrodes.

To carry out the method according to the invention, the suspension liquid 10 (carrier liquid) flows with particles 20 through the channel 30. The flow rate of the suspension liquid 10, which can be adjusted with a syringe pump is z. B. 300 microns / s. First, the particles 20 are preferably lined up with the dielectric line-up element 50. The funnel electrodes 51, 52 are operated, for example, with a high-frequency alternating voltage (f = 2 MHz, U = 20 V _pp ) to place the particles 20 on a flow path 11 in the middle of the channel 30 to focus. Alternatively, a hydrodynamic Aufreihelement be provided, in which the particles 20 are focused with additional enveloping streams.

After the alignment of the particles they reach the area of the electrodes 40. These are driven, for example, alternately with an alternating voltage and a direct voltage with a clock frequency in the range from 1 to 10 Hz (AC voltage: f = 2.5 MHz, U = 20 Vpp, DC voltage U = 50 V, duration t = 80 μs). By matching the voltage and frequency parameters of the high-frequency AC voltage to the flow rate and the adjustment of the DC parameters (pulse time, voltage and clock frequency), the smaller particles within a few seconds by a few 10 microns from the original flow path 11 in an adjacent flow path 12 (see 2), while the larger particles remain in the original flow path 11.

The potentials acting on the particles are schematically illustrated in FIG. For electrophoresis, a DC voltage field is generated which generates a potential P1 which drops transversely to the flow cross-section. Particles experience in the potential P1 an outward force (deflecting potential, deflection direction transverse to the flow direction). The high-frequency activation of the electrodes generates an opposite, inwardly directed, focusing potential profile P2a or P2b. The negative dielectrophoresis is based on a particle polarization, which has a stronger effect on the large particles than on the small particles. In the high-frequency field, therefore, the large particles 21 experience the potential P2a and the small particles 22 the shallower potential P2b. The superposition of the two cases with the focusing potential P1 gives the effective potentials Pa, Pb corresponding to the solid lines. While the deep potential P2a is hardly changed by the electrophoresis, a shift of the potential minimum from the center of the channel to the outside results for the flat potential P2b. For the large particles, the dielectrophoretic focusing forces are so great that they respectively compensate for the electrophoretic displacement, whereas for the small particles 21 this is not the case. Accordingly, the separate flow paths 11, 12 are formed. In the flow paths 11, 12 different flow velocities can be given. For example, with a laminar flow in the channel, the flow velocity near the channel wall is less than in the middle of the channel. According to the invention particles of different properties can thus be focused in areas with different flow velocities, which can improve the selectivity.

Analogous effects arise for particles with different relative dielectric constants or with different net charges, for example surface charges.

Experimentally, the separation was shown with a mixture of particles 20 comprising smaller 1 μm diameter particles ("Fluospheres" sulfate microspheres, Molecular Probes) and larger 4.5 μm diameter particles 22 (Polybeadpolystyrene, 17135, Polysciences) , As a suspension liquid Cytoconlösung I (Evotech Technologies GmbH, Hamburg, Germany) was used. Since the negative dielectrophoresis on the small particles is considerably weaker than on the large particles, the small particles can be pulled out of the middle flow path 11 by the electrophoretic force.

The electrode activation takes place, for example, according to the following scheme: Electrodes in FIG. 2 Phase of the high frequency alternating voltage Potential DC voltage 47 0 ° Dimensions 48 180 ° pulse 45 0 ° pulse 46 180 ° Dimensions

Alternatively, the electrode drive can take place, for example, according to the following scheme (rotating electric field): Electrodes in FIG. 2 Phase of the high frequency alternating voltage Potential DC voltage 47 0 ° Dimensions 48 90 ° pulse 45 270 ° pulse 46 180 ° Dimensions

To illustrate the combination of dielectrophoresis with other deflecting forces according to the invention, Figure 1 shows schematically a separator 40A (shown in phantom) The separator 40A provided in or outside the duct wall is, for example, a magnetic device for applying magnetic forces, a laser device for applying optical forces analogous to the principle the laser tweezer or a sound source for the exercise of mechanical forces z. B. by ultrasound.

Figure 4 shows features of modified embodiments of the invention. Notwithstanding Figure 1 may be provided that the flow path 11 is displaced from the center of the channel 30 to the outside, in which the potential minimum of the dielectrophoresis is shifted by appropriate asymmetric activation of the electrodes 40. Furthermore, it can be provided that the flow paths 11, 12 open into separate compartments 35, 36 of the channel 30, which are separated from one another by channel walls or (as illustrated) by an electric field barrier. The electric field barrier is created by at least one barrier on the electrode 60 which extends in the channel direction.

In the embodiment illustrated in FIG. 5, electrodes 41, 42 for electrophoresis are located in a channel 30 laterally on the channel walls 31, 32 and / or on the bottom surface 33, and at least one electrode 43 for dielectrophoresis is centrally located. The electrode 43 is provided in a manner known per se with an electrically insulating passivation layer 43a. The passivation layer 43a has two functions. First, it prevents field loss of the DC field for electrophoresis, second, it prevents permanent attachment and thus possibly associated denaturation of particles or electrochemical reactions at the electrodes. The electrodes 41, 42 and 43 are each connected to a DC voltage source and an AC voltage source.

Optionally, the channel edge can be realized by porous materials (eg hollow fibers). This makes it possible to impose additional external chemical gradients (eg a pH profile). Furthermore, the at least one electrode 43 and the electrodes 41, 42 may be arranged offset in the flow direction for electrophoresis.

For particle separation, flushed-in micro-objects (for example macromolecules) are pulled to the central electrode 43 by positive dielectrophoresis. Simultaneously or with alternating activation of the electrodes, the microobjects are drawn by electrophoresis to the edge of the channel 30. The separation is based on the principles described above of a differential effect of the combination of dielectrophoresis and electrophoresis on the different particles.

Alternatively, with the arrangement of FIG. 5, the following procedure can be realized. By dielectrophoresis, the particles are first collected at the central electrode 43. Subsequently, the lateral flow 10 is stopped by the channel 30 and carried out a separation of the micro-objects via electrophoresis. After the electrophoretic separation into different flow paths, the flow 10 is continued. The essential advantage of the interruption of the flow transport through the channel, which is optionally provided according to the invention during the electrophoresis, is that an increased selectivity of the electrophoresis can be achieved by the previously defined start conditions.

If a plurality of optionally passivated electrodes 43.1 to 43.5 are provided for dielectrophoresis, the structure shown in FIG. 6 results. In the channel 30 are three-dimensionally arranged on the side walls, the electrodes 41, 42 for electrophoresis and on the bottom surface of the electrodes 43.1 to 43.5 for dielectrophoresis (electrical leads not shown). On the top surface (not shown) are dielectrophoresis electrodes in the same number and arrangement as the electrodes 43.1 to 43.5. The electrodes 43.1 to 43.5 are subjected to signals which are phase-shifted between adjacent electrodes (for example 43.1, 43.2) by 180 ° and for in-phase electrodes (for example 43.1 and opposite electrode on the top surface) in phase are. For example, the particles 20 sparged with the flow 10 include two types, one type of which is not addressed by electrophoresis. The particles 20 first arrange themselves dielectrophoretically (negative dielectrophoresis) in the intermediate space of the electrodes standing one above the other (hidden in the top view). It is only when passing the electrophoretic field that the particles of one type are deflected, while the other type remains unaffected.

In the embodiment according to FIG. 7, too, many optionally passivated electrodes 43.1 to 43.11 for dielectrophoresis are arranged between the electrodes 41, 42 for electrophoresis. On the top surface (not shown) are dielectrophoresis electrodes in the same number and arrangement as the electrodes 43.1 to 43.11. The first dielectrophoresis electrode pair 43.1, 43.2 is provided with a dielectric line-up element 50 for increasing the selectivity. In contrast to the embodiments described above, in FIG. 7 the DC electrophoresis field (deflection direction) is aligned parallel to the flow direction of the liquid 10 (see arrow) through the compartment 30.

When controlling the dielectrophoresis electrode array with 180 ° phase shift between adjacent and opposite electrodes or with 90 ° phase shift, the particles 20 arrange between the electrodes (negative dielectrophoresis). The dielectrophoresis electrodes form a periodic i. A. asymmetric modulated potential to which the electrophoresis potential between the electrodes 41, 42 is superimposed. The asymmetric modulation of the dielectrophoresis fields means that alternately higher or lower field strengths are set between adjacent electrode strips of the array 43.1 to 43.11. The electrophoresis potential between the electrodes 41, 42 is not kept constant in time, but switched periodically or randomly. Leave it a highly sensitive separation according to the principle of the so-called Brown's ratchet ("Brownian ratchet" or Rüttelratsche, see H. Linke et al., Physikalische Blätter Bd. 56, No. 5, 2000, pp 45-47) realize. In Brown's ratchet, the migration speed of particles through Brownian motion is highly dependent on particle size. The separation takes place in different flow sections in the flow direction depending on the different migration velocities of the particles. A particular advantage of this procedure is that the separation over several adjustable parameters can be sensitively controlled by the superposition of Brownian motion, electrophoresis and dielectrophoresis. This embodiment of the invention is particularly suitable for molecular separation (eg, separation of DNA molecules or DNA fragments that are all negatively charged in a physiological environment).

With mixed population of differing charges (+/-), the input channel with the array element 50 should be centered on the array of dielectrophoresis electrodes so that different charge objects are electrophoretically moved in different directions. In planar structures can also realize asymmetric potential for positive dielectrophoresis, z. B. by applying asymmetric, so relative to the channel longitudinal direction, for example, different thick passivation layers.

FIG. 8 illustrates, like FIG. 2, a cross-sectional view of a fluidic microsystem 100 with four electrodes 45-48. With these electrodes, a focusing potential is generated whose potential minimum lies in the middle of the channel. At the same time, analogously to FIG. 3, a first electrical potential acting in the x direction is generated for an electrophoretic field effect, and additionally in the y direction a magnetic field gradient for forming a second deflecting potential. The magnetic field gradient is with a magnetic field generating element 49th formed, for example, a permanent magnet or a liquid-insulated, current-carrying conductor comprises. Notwithstanding the illustrated embodiment, the magnetic field generating element may be arranged at a distance from the channel.

As the particles travel in the z-direction through the channel, they experience a deflection in both x and y spatial directions, the magnitude of which depends on the dielectric and magnetic properties of the particles to be separated. This embodiment of the invention is used, for example, for the separation of latex-coated, superparamagnetic particles with the aim of obtaining fractions with high monodispersity.

The graph in FIG. 9 illustrates the dielectrophoretic force f _diel normalized to the respective volume, which acts on a particle in the alternating field as a function of the frequency of the alternating field. The simulation results refer to latex beads with diameters of 0.5 μm, 1 μm, 2 μm and 5 μm (curves from above) with a conductivity of 0.7 mS / m and DK = 3.5 in water. The symbolically illustrated electrodes are arranged analogously to FIG. 1 and are applied alternately or superimposed with a signal which contains frequency components below 100 kHz and above 1 MHz. The low-frequency and higher-frequency signal components are generated, for example, with equal amplitudes in time-quadratic mean, but different phase relationships illustrated in the image feeds. The higher-frequency signal focuses the particles by negative dielectrophoresis towards the center of the channel. In contrast, the low-frequency signal acts as a function of the particle size by positive or negative dielectrophoresis, which is superimposed on the focusing effect of the higher-frequency signal. As a result, the smaller particles are deflected to the top left, while the larger particles (eg 5 μm) collect on a diagonal line at the bottom right. Corresponding Particles of different sizes enter different flow paths within the flow through the channel.

The features of the invention disclosed in the foregoing description, drawings and claims may be significant to the realization of the invention in its various forms both individually and in combination.

Claims (33)

1. Method for separating particles (20, 21, 22) in a compartment (30) of a fluidic microsystem (100), comprising the steps:

   - displacing a liquid (10), in which particles (20, 21, 22) are suspended, in a predetermined flow direction through the compartment (30), and

   - generating a deflecting potential causing at least some of the particles (20, 21, 22) to be displaced in a deflected direction in relation to the liquid,

   characterised by the additional steps:

   - generating at least one focussing potential, such that, under the effect of high-frequency electrical fields, at least some of the particles are displaced by dielectrophoresis in an opposite direction to the deflection direction in relation to the liquid, and

   - directing particles which have different electric, magnetic or geometric properties into different flow regions (11, 12) in the liquid.
2. Method according to claim 1, in which the deflection direction differs from the flow direction and has a component at right angles to the flow direction.
3. Method according to claim 2, in which the deflection direction extends perpendicular to the flow direction towards at least one of the lateral walls of the compartment, the deflecting potential is generated by electric, magnetic, optical, thermal and/or mechanical forces, and the flow regions comprise flow paths (11, 12) which correspond to different potential minima which are formed for the respective particles by the superimposition of the deflecting and focussing potentials whilst passing through the compartment in a time average.
4. Method according to claim 3, in which the deflecting potential is formed by a direct voltage field, under the effect of which the particles are drawn by electrophoresis towards at least one of the lateral walls of the compartment (30).
5. Method according to claim 4, in which the particles include biological cells, at least some of which are lysed under the effect of the direct voltage field.
6. Method according to claim 3, in which the liquid (10) comprises a suspension of biological material which contains biological cells and cell components, separation of the cells from the cell components taking place under the effect of the direct voltage field.
7. Method according to claim 4, in which electrodes (40) are arranged on walls (31-34) of the compartment (30) and electrical fields are applied to them to generate the dielectrophoresis and the electrophoresis.
8. Method according to at least one of the preceding claims, in which the deflecting and focussing potentials are generated alternately in at least one portion of the compartment (30) or alternating geometrically in different successive portions of the compartment (30).
9. Method according to the preceding claims 5 and 6, in which the electrical fields comprise high-frequency alternating voltage components and direct voltage components which are generated simultaneously or alternately.
10. Method according to claim 7, in which a plurality of focussing potentials is generated between the two electrodes (41, 42) with an electrode array (43.1 to 43.11), the particles being directed onto the different flow paths (11, 12) according to their electric or geometric properties.
11. Method according to at least one of the preceding claims 2 to 9, in which the particles (20, 21, 22) are directed onto at least two separate flow paths (11, 12).
12. Method according to claim 11, in which the at least two flow paths (11, 12) open out into additional, separate compartments (35, 36) of the microsystem (100).
13. Method according to claim 12, in which the at least two flow paths (11, 12) open out into separate compartments (35, 36) of the microsystem (100) which are separated by compartment walls or electrical barriers (60).
14. Method according to claim 1, in which the deflection direction extends parallel to the flow direction and a plurality of focussing potentials are generated which are modulated asymmetrically parallel to the deflection direction and in which the particles pass through the deflecting potential at different speeds.
15. Method according to at least one of the preceding claims, in which the particles (20, 21, 22) flow in front of the electrodes past a dielectrophoretic or hydrodynamic alignment element (50).
16. Method according to at least one of the preceding claims, in which a pH gradient is generated in the channel (30).
17. Method according to claim 16, in which the pH gradient is generated by electrical direct voltage fields which are provided for the electrophoretic separation of the particles.
18. Method according to at least one of the preceding claims, in which detection of the particles takes place after the particles have been directed onto the different flow paths (11, 12).
19. Method according to at least one of the preceding claims, in which the deflecting and the focussing potential are formed by a plurality of superimposed direct current voltages of different frequencies.
20. Method according to at least one of the preceding claims, in which at least two deflecting potentials are generated which have different deflection directions.
21. Fluidic microsystem, comprising:

    - a least one compartment (30) through which a liquid having particles (20, 21, 22) flows in a predetermined flow direction, and

    - a first separating device for generating a deflecting potential causing the particles (20, 21, 22) to be displaced in a deflected direction,

    characterised by

    - a second separating device which has electrodes (40) for generating at least one focussing potential, such that the particles are displaced by dielectrophoresis in an opposite direction to the deflection direction.
22. Microsystem according to claim 21, in which the deflection direction differs from the flow direction.
23. Microsystem according to claim 21 or 22, in which the first separating device is set up for generating electric, magnetic, optical and/or mechanical forces.
24. Microsystem according to claim 23, in which the first separating device comprises electrophoresis electrodes, a magnetic field device, a laser or a source of ultrasound.
25. Microsystem according to at least one of the preceding claims 21 to 24, in which the first and second separating devices are arranged separately in different, successive portions of the compartment (30).
26. Microsystem according to claim 21, 23 or 25, in which the first and second separating devices form a common deflecting unit which includes the electrodes (40).
27. Microsystem according to claim 26, in which the deflecting unit can be triggered alternately with alternating voltage and with direct voltage.
28. Microsystem according to claim 24, in which there is arranged between the electrophoresis electrodes (41, 42) an electrode array (43.1 to 43.11) comprising electrode strips which can be triggered individually with high-frequency alternating voltages.
29. Microsystem according to claim 21, in which the deflection direction extends parallel to the flow direction.
30. Microsystem according to at least one of the preceding claims 21 to 29, in which the electrodes (40) are arranged on inner sides of the walls of the compartment (30).
31. Microsystem according to at least one of the preceding claims 21 to 30, in which compartment (30) opens out into separate compartments (35, 36) of the microsystem (100).
32. Microsystem according to claim 31, in which compartments (35, 36) of the microsystem (100) are separated by compartment walls or electrical barriers (60).
33. Microsystem according to at least one of the preceding claims 21 to 32, in which a dielectrophoretic or hydrodynamic alignment element (50) is arranged in the compartment (30) in front of the separating devices.

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